NF-xcexaB is a transcription factor that plays a critical role in promoting immunologic and proinflammatory responses. NF-xcexaB can be activated by a variety of cell surface receptors including members of the TNF receptor family and the Toll-like receptor (TLR) family (Ghosh, S., et al. (1998) Annu. Rev. Immunol., 16:225). Although different receptors often employ distinct combinations of intracellular adapter proteins to initiate the NF-xcexaB activation process, the signals converge downstream into a common pathway that leads to phosphorylation and degradation of the NF-xcexaB inhibitory protein IxcexaB. The precise step where signal integration occurs remains unclear (Ghosh, S., et al. (1998) Annu. Rev. Immunol., 16:225; Karin, M., (1999) J. Biol. Chem., 274:27339).
One element common to many NF-xcexaB activation pathways is the IxcexaB kinase (IKK) complex, a cytokine-activated, multicomponent complex that specifically phosphorylates IxcexaB on two serine residues. The IKK complex consists of two kinase subunits (IKKxcex1 and IKKxcex2) and one regulatory subunit (NEMO or IKKxcex3) and is thought to be activated by phosphorylation of activation loop serines in IKKxcex1 and IKKxcex2 (Regnier, C., et al. (1997) Cell, 90:373; DiDonato, J., et al. (1997) Nature, 388:548; Mercurio, F., et al. (1997) Science, 278:860; Woronicz, J., et al. (1997) Science, 278:866; Zandi, E., et al. (1997) Cell, 91:243; Yamaoka, S., et al. (1998) Cell, 93:1231; Rothwarf, D., et al. (1998) Nature, 395:297).
NIK (NF-xcexaB inducing kinase), a MAP3K-like kinase, has been reported to be the kinase responsible for IxcexaB activation by many different stimuli (Malinin, N., et al. (1997) Nature, 385:540; Song, H., et al. (1997) Proc. Natl. Acad. Sci. U.S.A., 94:9792). A role for NIK in the NF-xcexaB signaling pathway has been inferred based on various observations, including that overexpression of NIK induces NF-xcexaB activation in cells in a ligand-independent manner and that overexpression of a kinase inactive, NIK mutant inhibits NF-xcexaB activation by a variety of ligands (Malinin, N., et al. (1997) Nature, 385:540; Song, H., et al. (1997) Proc. Natl. Acad Sci. U.S.A., 94:9792).
Recently a mutant strain of mice (alymphoplasia mice, aly/aly mice) has been identified that produce a mutant form of NIK. The abnormal protein is catalytically active but contains a single amino acid substitution near the carboxy-terminus in a region that can interact with the TRAF family of adapter proteins (Shinkura, R., et al. (1999) Nat. Genet., 22:74). These mice display defective lymph node (LN) development, lack IgA and have significantly reduced serum levels of IgM and lgG (Miyawaki, S., et al. (1994) Eur. J. Immunol., 24:429). Embryonic fibroblasts (MEFs) derived from these mice activate normal levels of NF-xcexaB in response to TNF, and the mice show unaltered LPS-induced shock responses (Shinkura, R., et al. (1999) Nat. Genet., 22:74). However, due to the presence of a catalytically-active mutant NIK protein in the aly/aly mouse, it is not possible to draw firm conclusions based on these findings about the role of NIK in either NF-xcexaB signaling or induction of cytokine-dependent biologic responses.
The integrity of the skeleton is maintained by a balance between osteoclasts, which promote bone resorption, and osteoblasts, which promote bone production. Under some circumstances, such as with age or under certain medical conditions, the balance of bone resorption and rebuilding can be disrupted, and bone density can be altered, often having deleterious effects. For example, a loss of bone density can result in conditions such as osteoporosis, which is characterized by low bone mass and structural deterioration of bone tissue, resulting in bone fragility and an increased susceptibility to fractures. In the U.S. today, 10 million individuals are affected by osteoporosis, and an additional 18 million more have low bone mass.
Clearly, there is a need in the art for better tools with which to study the signal transduction pathway leading to NF-xcexaB activation in different cell types. In addition, there is a great need for new methods for treating and preventing osteoporosis and other disorders, such as by inhibiting the activity of osteoclasts. The present invention addresses these and other needs.
The present invention provides transgenic non-human mammals and cells having one or more structurally and functionally-disrupted NIK alleles. In addition, the present invention provides methods for making such transgenic mammals and cells, as well as methods for determining the effect of a compound on an animal or cell that lacks NIK function. The present invention also provides methods for identifying compounds useful in the inhibition of osteoclastogenesis in a mammal, for example to treat osteoporosis or other conditions. Also provided are methods of modulating the extent of osteoclastogenesis in a cell or a mammal using NIK modulators.
In one aspect, therefore, the present invention provides a transgenic non-human mammal having at least one structurally and functionally disrupted NIK allele, wherein said NIK allele is disrupted by insertion of a transgene within a NIK locus within the genome of said mammal.
In one embodiment, the mammal is homozygous for the disrupted NIK allele. In another embodiment, the mammal shows one or more phenotypic effects of the disrupted NIK allele selected from the group consisting of impaired lymph node development, absence of Peyer""s Patches, disrupted lymphoid architecture, decreased IgA levels, decreased IgG2b levels, impaired NF-xcexaB activation in response to anti-LTxcex2R and impaired osteoclastogenesis in response to OPGL. In another embodiment, the transgene is inserted into the NIK locus by homologous recombination, and the insertion removes at least a portion of an endogenous NIK gene within the genome of said mammal, the portion comprising at least I kb, and including at least 40 codons, of the endogenous NIK gene. In another embodiment, the insertion of the transgene results in the replacement of a portion of the endogenous NIK gene with a nucleic acid sequence encoding a selectable marker. In another embodiment, the selectable marker is a neomycin-resistance gene. In another embodiment, the mammal is a mouse.
In another aspect, the present invention provides a cell derived from any of the above-described transgenic mammals.
In another aspect, the present invention provides an animal model for determining the effect of a test agent on a host deficient in NIK function, the model comprising any of the above-described transgenic mammals.
In another aspect, the present invention provides a method of making a transgenic non-human animal comprising at least one structurally and functionally disrupted NIK allele, the method comprising: (i) transfecting a plurality of embryonic stem cells with a nucleic acid comprising a NIK gene that is disrupted by insertion of a selectable marker, (ii) selecting for transgenic embryonic stem cells that have incorporated the nucleic acid into their genome; (iii) introducing at least one of the transgenic embryonic stem cells into an embryo to produce a chimeric mammal comprising at least one of the transgenic embryonic stem cells; and (iv) breeding the chimeric mammal with a wild type mammal to obtain F1 progeny that are heterozygous for the disrupted NIK gene.
In one embodiment, the animal is a mouse.
In another embodiment, the present invention provides a method for determining the effect of a test agent on a mammal deficient in NIK function, the method comprising: (i) contacting a transgenic non-human mammal having at least one disrupted NIK allele with said test agent; and (ii) detecting the presence or absence of a functional effect of the test agent in the mammal.
In one embodiment, the mammal is a mouse. In another embodiment, the mammal is homozygous for a structurally and functionally disrupted NIK allele. In another embodiment, the transgenic non-human mammal is generated using any of the abovedescribed methods.
In another aspect, the present invention provides a method of identifying a compound useful in the treatment of osteoporosis in a mammal, the method comprising: (i) contacting a NIK polypeptide with a test compound; and (ii) detecting the functional effect of the test compound on the NIK polypeptide.
In one embodiment, the functional effect comprises an inhibition of NIK kinase activity. In another embodiment, the NIK polypeptide is present within a cell. In another embodiment, the functional effect comprises detecting osteoclastogenesis of the cell.
In another aspect, the present invention provides a method of inhibiting osteoclastogenesis in a cell, the method comprising contacting the cell with a compound that inhibits the activity or expression of NIK in the cell.
In one embodiment, the compound is an antisense oligonucleotide. In another embodiment, the compound is a small molecule inhibitor of NIK kinase activity, or an inhibitor of NIK interaction with receptor or adaptor proteins in cells. In another embodiment, the cell is present within a mammal, and the compound is administered systemically or locally to the mammal. In another embodiment, the compound is identified using any of the above-described methods.